The main objective of this project is to characterize the potential role of oxidative stress in regulating the extensive proliferative potential of acute myeloid leukemia stem cells. Acute myeloid leukemia (AML) is initiated by cancer causing mutations that transform normal hematopoietic stem and progenitor cells into phenotypically heterogeneous population of malignant cells. Based on functional analogies with normal stem cells, the subset of AML cells with the unique developmental potential to drive and sustain the disease are dubbed leukemic stem cells (LSC). LSCs have distinct, evolved biological properties that render them resistant to standard clinical drugs. As a result, understanding the defining molecular properties of LSCs is deemed critical for the development of novel and improved therapeutic strategies. Among the many factors known to regulate the biology of normal stem cells and tumor cells, one of the best characterized is the level of intrinsic cellular Reactive Oxygen Species (ROS). Thus, a central premise of our work is the notion that molecular mechanisms regulating the levels of ROS in AML, particularly in LSCs, represent crucial therapeutic targets. With this in mind, we conducted a series of experiments to dissect the role of ROS in regulating the fundamental cellular properties of leukemic stem cells. Our studies revealed that based on the level of ROS, individual primary human AMLs could be segregated into two biologically distinct sub-populations (ROS-low and ROS-high cells). Interestingly, in murine xenograft assays, the leukemia initiating potential is primarily contained in the ROS-low compartment, suggesting the preferential localization of leukemic stem cells in the lower oxidative state. Gene expression and biochemical analysis performed on purified subpopulations identified the expression of a ROS producing enzyme complex in leukemic stem cells. Importantly, pharmacologic and genetic inhibition of this complex reduced the level of ROS and limited cell proliferation and survival. This proposal will test the hypothesis that enzymatic sources of ROS are important for the genesis and perpetuation of leukemic stem cells and that inhibiting these sources will be a viable therapeutic option for treating AML patients. The goals of this project are two-fold. 1) To investigate whether enzymatic sources of ROS are required for the transformation and maintenance of leukemic stem cells. 2) To evaluate the therapeutic potential of targeting oxidative metabolism in a preclinical mouse model of AML. To achieve these goals, we will utilize a genetically defined murine model of leukemia and xenograft models of primary human AML samples from patient biopsies in immune compromised mice. This proposal will help the National Cancer Institute fulfill their mission to support research projects for the treatment of cancer and to support education and training in fundamental sciences and clinical disciplines relating to cancer.